The ability to survive acute lung injury correlates with the ability to maintain alveolar fluid clearance. Alveolar fluid balance is maintained, in part, by epithelial tight junctions which create a permeability barrier to free diffusion between cells. Transmembrane proteins in the claudin family are a major functional component of tight junctions. There are roughly two dozen mammalian claudins and alveolar epithelial cells use differential expression of multiple claudins to regulate the tight junction barrier. Recent work suggests that alveolar epithelial cells also regulate the process of claudin assembly into tight junctions. However, little is known about the mechanisms that regulate claudin trafficking and assembly in general and by alveolar epithelial cells in particular. Also, the rules governing how different claudins can intermix have not been identified. To define the mechanisms which underlie claudin trafficking, we will use a series of cell and molecular approaches to: 1) define roles for EMP-family proteins in regulating claudin traffic, assembly and barrier function, 2) define intracellular sites for claudin oligomerization and determine motifs regulating heteromeric claudin interactions and 3) identify claudin motifs which determine extracellular head-to-head binding specificity. By defining control points that are critical to regulate alveolar barrier function, these studies are expected to provide a molecular and cell biological basis to help design strategies to prevent the alveolar leak that accompanies lung injury.